There is currently a need for rapid and cheap polynucleotide (e.g. DNA or RNA) sequencing and identification technologies across a wide range of applications. Existing technologies are slow and expensive mainly because they rely on amplification techniques to produce large volumes of polynucleotide and require a high quantity of specialist fluorescent chemicals for signal detection.
Transmembrane pores (nanopores) have great potential as direct, electrical biosensors for polymers and a variety of small molecules. In particular, recent focus has been given to nanopores as a potential DNA sequencing technology.
When a potential is applied across a nanopore, there is a change in the current flow when an analyte, such as a nucleotide, resides transiently in the barrel for a certain period of time. Nanopore detection of the nucleotide gives a current change of known signature and duration. In the strand sequencing method, a single polynucleotide strand is passed through the pore and the identities of the nucleotides are derived. Strand sequencing can involve the use of a polynucleotide binding protein to control the movement of the polynucleotide through the pore.
It has previously been demonstrated that ultra low concentration analyte delivery can be achieved by coupling the analyte to a membrane in which the relevant detector is present. This lowers by several orders of magnitude the amount of analyte required in order to be detected (WO 2012/164270).
It has also been shown that double stranded polynucleotides can be effectively characterised using strand sequencing if they are modified to include a Y adaptor (a double stranded stem and two non-complementary arms) containing a leader sequence and a hairpin loop adaptor (WO 2013/014451). It is preferred that that Y adaptor containing the leader sequence is attached to one end of the polynucleotide and the hairpin loop adaptor is attached to the other end. The leader sequence preferentially threads into the nanopore and the hairpin loop connecting the two strands of the polynucleotide allows both strands to be investigated as the polynucleotide unzips and moves through the pore. This is advantageous because it doubles the amount of information obtained from a single double stranded polynucleotide. Moreover, because the sequences in the two strands are complementary, the information from the two strands can be combined informatically. This mechanism provides an orthogonal proof-reading capability that provides higher confidence observations. When Y adaptors and hairpin loops are used together in this way, the Y adaptor typically contains an anchor which couples the polynucleotide to the membrane containing the nanopore. In some instances, double stranded polynucleotides having Y adaptors at both ends are produced in the sample preparation. The presence of the leader sequence and the anchor in the Y adaptors means that the system is typically biased towards characterising these polynucleotides. However, the lack of the hairpin loop linking the two strands in such polynucleotides means that only one strand is investigated.